Personalized medicine" is a new clinical paradigm where a specific cancer therapeutic ("smart drug") is prescribed based upon the correlation of proteomic profiles from diseased tissues with patient's response to drug therapy. Many of these new "smart drug" therapies specifically target growth factor receptors and inhibit signal transduction in cancer cells. The overall goal of this proposal is to develop a diagnostic tool based on our innovative Layer Protein Screening (LPS). This tool will be used for profiling of Akt in order to monitor the response of breast cancer patients to treatment with inhibitors of growth factor signaling. Akt is a major anti-apoptotic molecule downstream of growth factor receptors [Her-2/neu, epidermal growth factor receptor (EGFR) and insulin growth factor receptor (IGFR)]. Thus, Akt and its downstream targets are potentially good monitors of the efficacy of growth factor inhibitors. Furthermore, there is a critical need for a practical tool that can generate expression profiles for Akt and related proteins from small amounts of patient tissue samples. 20/20 Gene Systems, Inc. has developed LPS as a quantitative, sensitive, comprehensive analysis of protein expression profiles in human cells and tissues. This technology allows simultaneous evaluation of up to ten different proteins from the same tissue or cell sample. It is compatible with analysis of essentially any protein, it is robust, easy to standardize, is amenable to high throughput, and preserves tissue morphology. We hypothesize that LPS will provide the ability for comprehensive and quantitative analysis of status of Akt in tissue samples from breast cancer patients treated with inhibitors of growth factor signaling. We expect that experiments outlined in this proposal (Phase I) will allow us to develop a prototype of a tissue-based LPS assay for comprehensive and quantitative analysis of Akt status in normal and tumor human breast tissues. The long term goal is to produce the "LPS-Akt Assay Kit" that will be developed during Phase II. To establish proof of principle in Phase I, we will test the ability of the LPS to profile the activity of EGFR, Akt and PARP in: 1) Human mammary epithelial cell line samples (by cross-validating LPS-tissue blot with traditional immunoblotting), 2) Tissue sections of human breast cancer (with adjacent normal tissue) by comparing LPS tissue blot to immunohistochemistry.